Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell disorders with frequent transformation to acute myeloid leukemia (AML) and high mortality. The most common cytogenetic abnormality is a deletion within chromosome 5q (del(5q)). Patients with del(5q) MDS exhibit peripheral blood (PB) cytopenias, some attributed to increased p53 activity resulting in the apoptosis of erythroid progenitors and decline of hematopoietic stem/progenitor cell (HSPC) function. To understand these phenotypes, individual gene functions must be elucidated. TRAF-interacting protein with a forkhead-associated domain B (TIFAB) is deleted in nearly all cases of aggressive del(5q) MDS/AML. We have shown that TIFAB-deficient mice exhibit increased innate immune/Toll-like receptor signaling through increased levels of TRAF6 and TRAF6 has shown to be overexpressed in MDS and AML. However, TRAF6 overexpression alone is not sufficient for leukemogenesis, suggesting that an opposing pathway is active in preventing transformation. In parallel, our preliminary data indicate that TIFAB-deficient mice exhibit increased levels of p53 in the bone marrow, aberrant p53 gene signatures, and hypersensitivity to cellular stress, which impairs HSPC growth. Conversely, overexpression of TIFAB limits transcript expression of p53 target gene, P21, implicating TIFAB as a negative regulator of p53. To understand the molecular mechanism of TIFAB, we performed mass-spectrometry analysis of TIFAB-interacting proteins and identified ubiquitin-specific peptidase 15 (USP15) as the top hit. Importantly, USP15 has been shown to negatively regulate p53 via deubiquitination and destabilization of MDM2. Taken together, our findings support a model that loss of TIFAB activates parallel, opposing pathways that both promote oncogenesis via increased innate immune signaling, yet prevent transformation by limiting USP15 activity and thus MDM2-dependent regulation of p53. The goal of this proposal is to determine on a molecular level how the loss of TIFAB contributes to MDS pathogenesis and permits leukemic transformation. To address this, we have proposed two aims: Aim 1 will address the molecular mechanism of the TIFAB- USP15 complex and reveal the cellular consequences of its loss. Aim 2 will explore the consequences of TIFAB loss coupled with consecutive loss of p53. By identifying the molecular mechanism of TIFAB, the biological function of the TIFAB-USP15 complex, and how its loss cooperates with p53 inactivation, we will define a causal link between the del(5q) aberration and the pathogenesis of MDS/AML. Investigation of the function of TIFAB is warranted for the purpose of exploring new therapeutic targets, of which there are currently few.